Abstract:

Cancer metastasis is the toughest problem in cancer therapy. Once tumor cells have the ability of surrounding cell invasion, the survival
rate of patient with tumor disease will be reduced. Hence, prevent or reduce the cancer metastasis will can increase the patients’ survival
rate. Apigenin has been demonstrated optimal effect in cancer prevention and reduce cancer metastasis, including breast cancer. In this
study, we verify low dose (≤5 μg/ml) apigenin exposure 3 h would not influence the MCF-7 cells viability but can decrease its FAK
signal activation to reduce matrix metalloproteinases (MMP-2 and MMP-9) expression. Moreover, low dose apigenin treated in MCF-7
cells could reduce the cell mobility through the Rho GTPases (Ras, Rac-1, cdc-42, and RhoA) downregulation to cause cytoskeleton
remodeling. Presented results have demonstrated the role of apigenin on breast cancer metastasis is associated with FAK signal pathway
inhibition. The capacity of low dose apigenin treated in breast cancer for short time has been clarified here. And thus may find widespread
application in clinical therapy as an anti-metastatic medicament.

Introduction:

Breast cancer is the common cancer of women and the ranking
5th leading cause of death worldwide [1]. The risk factor of breast
cancer development including, obesity, drinking alcohol, early
age at first menstruation, having children late or not at all, older
age, etc. About 5–10% of cases are genomic alteration, including
BRCA1 and BRCA2 mutation [2]. Surgery is the major method to
remove the tumor. Chemotherapy, radiation therapy and hormone
therapy are often used to reduce the recurrence. The majority of
early-stage breast cancer patients was undergo breast conserving
surgery and then receive adjuvant treatment. The 5-years survival
of early-stage breast cancer is 98.6%. While in late-stage, often
associated with metastasis, these therapies could not achieve the
optimal treatment and cause the survival rate declines to 24.3% [3].
Furthermore, once breast cancer with metastasis will cause poor
outcome. Therefore, inhibition of breast cancer metastasis can
extend the patients' survival.

Cancer metastasis is a complex process, the local tumor cells
migrate to distant site by the bloodstream, the lymphatic system,
or by direct extension. Among them, the epithelial-mesenchymal
transition (EMT) is the important characteristics. EMT is the
conserved developmental process in the evolution [4], and it also
plays pivotal role in cancer metastasis. Cytokines and growth
factors, such as transforming growth factor β (TGF-β) are related
with EMT dysregulation during the process of malignant tumor
[5]. In the EMT process, the cell-cell junction would be breakdown
by E-cadherin reducing, and the Rho GTPase function also be
modulated, loss of cell polarity ensues, a dramatic reorganization
of the actin cytoskeleton to enhance cell motile characteristics [5-7].
Once cancer metastasis, the survival rate of cancer patients would
be reduced. But, the molecular mechanism of cancer metastasis
happen is unclear. Therefore, in clinical, inhibition of cancer
metastasis is the crucial issues to prolong the life of tumor diseases
patients.

Cell culture and apigenin treatment

Human breast cancer cells, including MCF-7, MDA-MB-231, and
H184B5F5/M10 were maintained in DMEM medium containing
with 10% fetal calf serum and antibiotics (100 U/ml of penicillin
and 100 mg/ml of streptomycin). All cells were cultured at 37°C in
a humidified atmosphere of 5% CO2-95% air. The stock solution
of apigenin was dissolved in dimethylsulfoxide (DMSO) and
sterilized by filtration through 0.2 μm disc filters. Suitable amounts
of stock solution (10 mg/ml in DMSO) of apigenin were added
into the cultured medium to achieve the indicated concentrations
(final DMSO concentration was < 0.2%).

Assessment of cell viability (MTT assay) :

To explore the effect of apigenin on breast cancer cell viability, the
various concentration (0, 1, 2.5, 5, 7.5, 15, 30, and 45 μg/ml) were
respectively treated in MCF-7, MDA-MB-231, and H184B5F5/
M10 cell lines for 24 and 48 h. At the end of the assay period, cell
viability was measured by MTT [3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl-tetrazolium bromide] assay. Each concentration was
repeated three times. After the exposure period, the medium was
removed and followed by washing of the cells with PBS. Then,
the medium was changed and incubated with MTT solution (5 mg/
ml) per well for 4 h. The medium was removed, and formazan was
solubilized in isopropanol and measured spectrophotometrically at
563 nm. The percentage of viable cells was estimated by comparing
with the untreated control cells (0 μg/ml).

Boyden chamber invasion and migration assay

Matrigel (BD Transduction Laboratories, San Jose, CA) was be
diluted to 200 μg/ml with cold filtered distilled water, and then
added to the upper chamber with 8µm pore polycarbonate filter.
And then the treated MCF-7 cells (1×105 cells/well) were mixture
with serum free medium to add into the upper chamber. Culture
medium containing with 10% fetal bovine serum was applied to the
lower chamber as chemoattractant. The chamber was incubated for
8h at 37°C. At the end of incubation, the cells in the upper surface
of the membrane were carefully removed with a cotton swab. The
invasive cells were fixed by 3% methanol and then stained by 5%
Giemsa solution. The invading cells on the lower surface of the
membrane filter were counted with a light microscope. The data
are presented as the average number of cells attached to the bottom
surface from five random fields. Each experiment was carried out
in triplicate.
To measure the migrative ability of MCF-7 cells, cells were
seeded on the upper surface of the filter inserts with 8-μm pore
polycarbonate filters that were not coated with matrigel. The
migrative cells were treated and measured as described in the
invasion assay.

Wound-healing assay

To determine cell motility determination, MCF-7 cells (1×105cells/
ml) were seeded in 6-well tissue culture plate and grown to 80-
90% confluence. After aspirating the medium, the center of the cell
monolayers was scraped with a sterile micropipette tip to create
a denuded zone (gap) of constant width. Subsequently, cellular
debris was washed with PBS, and the cells were exposed to various
concentrations of apigenin (0, 1, 2.5, and 5 μg/ml). The wound
closure was monitored and photographed at 0, 6, 12, 24, and 48 h
with an Olympus CKX-41 inverted microscope and an Olympus
E-410 camera. To quantify the migrated cells, pictures of the initial
wounded monolayers were compared with the corresponding
pictures of cells at the end incubation. Artificial lines fitting the
cutting edges were drawn on pictures of the original wounds and
overlaid on the pictures of cultures after incubation. Migrated cells
across the white lines were counted in six random fields from each
triplicate treatment, and data are presented as mean ± SD.

Immunofluorescence assay

To determine the effect of apigenin on EMT and cytoskeleton
change, MCF-7 cells (4×105 cells/well) were plated in six-well
plates and grown for 24 h and then incubated in the different
concentrations of apigenin (0, 1, 2.5, and 5 μg/ml) for 24 h. After
the exposure period, the cells were washed with Ca2+/Mg2+
free PBS after cell medium removed. Cells were fixed with 4%
paraformaldehyde in PBS for 15 min and incubated with 0.5%
Triton X-100 in PBS for 5 min. Cells were incubated with 1% bovine serum albumin in 0.5% PBST at room temperature for 1 h.
And then add the primary antibody or phallodin-PE (Invitrogen,
Karlsruhe, Germany) to incubate at room temperature for 1 h.
The fluorescent images were visualized with a BX51 fluorescence
microscope (Olympus, Tokyo, Japan).

Analysis of MMP-2/9 activity by gelatin zymography

MCF-7 cells (4×105 cells/ml) were seeded into the 10 cm culture
dish and then incubated in the different concentrations of apigenin
(0, 1, 2.5, and 5 μg/ml) for 24 h. Subsequently, the conditioned
medium was collected and gelatin zymography was performed
to examine the activities of MMP-2 and MMP-9. Samples were
mixed with loading buffer and electrophoresed on 8% SDSpolyacrylamide
gel containing 0.1% gelatin. Electrophoresis was
performed at 140 and 110 V for 3 h. Gels were then washed twice in
zymography washing buffer (2.5% Triton X-100 in double-distilled
H2O) at room temperature to remove SDS, followed by incubation
at 37°C for 12-16 h in zymography reaction buffer (40 mM TrisHCl,
10 mM CaCl2, 0.02% NaN3), stained with Coomassie blue
R-250 (0.125% Comassie blue R-250, 0.1% amino black, 50%
methanol, 10% acetic acid) for 1 h and destained with destaining
solution (20% methanol, 10% acetic acid, 70% double-distilled
H2O). Nonstaining bands representing the levels of the latent
forms of MMP-2 and MMP-9 were quantified by densitometer
measurement using a digital imaging analysis system.

Reverse transcriptase polymerase chain reaction (RT-PCR)

Total RNA was isolated from MCF-7 cells using the total RNA
Extraction Midiprep System (Viogene BioTek, Taiwan). Total RNA
(2 μg) was transcribed to 20 μl cDNA with 1 μl deoxynucleotide
triphosphate (dNTP; dNTP set consists of 2.5 mM aqueous
solutions at pH 7.0 of each of dATP, dCTP, dGTP, and dTTP), 1 μl
Oligo dT (10 pmol/ml), 1 μl RTase (200 U), 1 μl RNase inhibitor
and 5× reaction buffer. The appropriate primers (sense of MMP-2,
5’-GGCCCTGTCACTCCTGAGAT -3’, nt 1337-1356; antisense
of MMP-2, 5’-GGCATCC AGGTTATCGGGGA-3’, nt 2026-
2007; sense of MMP-9, 5’-AGGCCTCTACAGAGTCTT TG-3’,
nt 1201-1220; antisense of MMP-9 were used for polymerase chain
reaction (PCR) amplifications. PCR was performed with Platinum
Taq polymerase (Invitrogen, San Diego, CA, USA) under the
following conditions: 30 cycles of 94°C for 1 min, 59°C (MMP-2)
or 60°C for 1 min (MMP-9 and β-actin), 72°C for 1 min followed
by 10 min at 72°C. The final products were electrophoresis in 2%
agarose gel and then detected through ethidium bromide staining.

Western blotting analysis

The denatured samples (50 μg extracted protein) were resolved on
10-12% SDS-PAGE gels. The proteins were then transferred onto
nitrocellulose membranes. Non-specific binding of the membranes
was blocked with Tris-buffered saline (TBS) containing 1% (w/v)
nonfat dry milk and 0.1% (v/v) Tween-20 (TBST) for more than
2 h. Membranes were washed with TBST three times for 10 min
and incubated with an appropriate dilution of specific primary
antibodies in TBST overnight at 4°C. Subsequently, membranes
were washed with TBST and incubated with appropriate secondary
antibodies (horseradish peroxidase-conjugated goat antimouse or
antirabbit IgG) for 1 h. After washing the membranes three times
for 10 min in TBST, the bands detection were revealed by enhanced
chemiluminescence using ECL Western blotting detection
reagents and exposed ECL hyperfilm in FUJFILM Las-3000 mini
(Tokyo, Japan). Then proteins were quantitatively determined by
densitometry using FUJFILM-Multi Gauge V3.0 software.

Statistical analysis

Data were expressed as means ± standard deviation of three
independent experiments and statistical analysis was obtained
using a Student’s t-test. All statistical analyses of data were
performed using Sigma Plot 2001 software (Systat Software Inc.,
San Jose, Calif., U.S.A.). Significant differences were established
at P ≤ 0.05.

To explore the optimal concentration of apigenin to influence
breast cancer cell, the MTT assay is used here. The concentration
of apigenin including 0, 1, 2.5, 5, 7.5, 15, 30, and 45 μg/ml were
respectively treated in MCF-7, MDA-MB-231 and H184B5F5/
M10 breast cancer cell lines. After treatment for 24 or 48 h, we
noticed low dose (≤5 μg/ml) apigenin no toxicity in MCF-7 cells,
and the cell viability can achieve 80%. However, once the dose of
apigenin more than 5 μg/ml, the cell viability dramatic decrease.
But, this phenomenon could not observe in MDA-MB-231 cells
(Fig. 1a). In pathologic characteristics, the MDA-MB-231 cell is
basal type breast cancer cell and associated with poor prognosis,
in turn, MCF-7 cell is a luminal type breast cancer have better
5-years survival rate. Imply the low dose apigenin treatment in
luminal type breast cancer can achieve the optimal treat efficiency
more than in basal type.

Next, we explore the role of apigenin in MCF-7 cells under the low
dose treatment. Apigenin has been shown can inhibit invasion and migration in colorectal cancer [20]. In cell migration assay and cell
invasion assay, we observe the phenomenon of cell migration was
reduced and the invasive cells were significantly decreased (Fig. 1b
and Fig. 1c). These results suggest that the optimal concentration
of apigenin in breast cancer cell treatment is ≤5 μg/ml to reduce
cell migration and invasion.

Fig. 1 Low dose apigenin treatment reduces cell migration and invasion. (a) MTT assay to determine cell viability of MCF-7 and
MDA-MB-231 cells, and found the low dose (0-5 μg/ml) apigenin no effect cell viability in MCF-7 cells as compared with normal
cell line, H184B5F5/M10. It is worth mentioning that MDA-MB-231 cells viability would not decrease in the apigenin presence. That
is to say, low dose apigenin treatment achieves optimal effect is manifested in MCF-7 cells rather than MDA-MB-231 cells. (b) The
time-dependant manner of MCF-7 cells migration can be inhibited by the low dose apigenin exposure 6, 12, 24 or 48 h to enter the tip
scratch area. (c) The dose-dependant manner of MCF-7 cells invasion significantly reduced by the apigenin exposure 24 or 48 h. *P as
p<0.05; **P as p<0.01; ***P as p<0.001.

Low dose apigenin reduce the MMP-2/9 and EMT expressions

Known the low dose apigenin can reduce the MCF-7 cells
migration and invasion during 24 h treatment, we next to verify
the MMP-2 and MMP-9 expression under 3 h exposure of low
dose apigenin. In the zymography results, we observed the MMP-
2 and MMP-9 decreasing in MCF-7 cells after low dose apigenin
exposure (Fig. 2a). In the meantime, we noticed the RNA level
of MMP-2 and MMP-9 were not change (Fig. 2b), suggest that
exposure 3h of low dose apigenin is influence protein expression
rather than genomic change. The results imply the apigenin can
inhibit breast cancer metastasis through matrix metalloproteinase
and plasminogen activator down regulation.

We next try to clarify the EMT change whether manifested in MCF-
7 cells after exposure 3 h of low dose apigenin. After fluorescence
microscopy imaging, we noticed the E-cadherin increase and the
fibronectin decrease in MCF-7 cells under the low dose apigenin
exposure (Fig. 2c). These results imply the low dose apigenin
can inhibit MCF-7 cells metastasis through MMP-2 and MMP-9
down regulation. In addition, it also can down-regulate EMT after
apigenin exposure 3 h.

Apigenin inhibit the MCF-7 cells metastasis through FAK
signals

To clarify the molecule mechanism of breast cancer metastasis
could be inhibited by low dose apigenin exposure 3 h, we use
the western blot experiment to demonstrate. Literatures indicate
apigenin can regulate PI3K/Akt signal pathway to inhibit breast
cancer cell metastasis [19], and promote apoptosis via a caspasedependent
mechanism [21]. The PI3K/Akt signal is the down stream
of Focal Adhesion Kinase (FAK). FAK transmits signal to the
cell interior through the candidate proteins phosphorylation and
dephosphorylation to cause MMP-9 decreased while metastasis
reduced [22]. However, we noticed the MMP-2, and MMP-9
were decreased after low dose apigenin exposure for 3 h, imply
FAK signal is the major pathway to regulate breast cancer cell also slightly decreased. In the meantime, the nuclear NFκB, c-Fos,
and c-Jun were downregulated (Fig. 3b), illustrate the FAK signal
can be inhibited by apigenin exposure 3 h.

Fig. 2 Low dose apigenin can influence the matrix metalloproteinase expression and EMT. (a) Galetin zymography assay was used
to determine the MMP-2 and MMP-9 expression under low dose apigenin exposure 3 h. We noticed the MMP-2 and MMP-9 were
be decreased by low dose apigenin especially in the concentration of 5 ug/ml. (b) RT-PCR data indicated that the MMP-2 and MMP-
9 expression decrease in low dose apigenin exposure 3 h is not involved in genomic change. (c) Fluorescence images show that the
dose-dependent manner of MCF-7 cells treated with low dose apigenin could cause E-cadherin slight increase and fibronectin decrease.
Scale=50 μm.

Apigenin can regulate the cytoskeleton change to inhibit the
MCF-7 cells metastasis

As described above, low dose apigenin exposure 3 h down-regulate
MMP-2 and MMP-9 through FAK inhibition. On the other hand,
we also noticed cytoskeleton regulator influence in the low dose
apigenin exposure 3 h. The Ras, Rac-1, Cdc42, and RhoA were
the downstream of FAK associated with cytoskeleton remodeling.
Known the Ras and Rac-1 are related with lamellipodia, the Cdc42
is a regulator in filopodia, and the RhoA is associated with stress
fiber formation. Here, we observed the Ras and Rac-1 obviously
down regulation after low dose apigenin exposure 3 h as compared
with Cdc-42 and RhoA (Fig. 4a). After fluorescence microscopy
imaging, we also observed the lamellipodia was not obviously
express in MCF-7 cells after low dose apigenin exposure 3 h (Fig.
4b). The results suggest that apigenin inhibit breast cancer cell
metastasis is through FAK signal down regulation to cause matrix
metalloproteinase decreasing and cytoskeleton remodeling (Fig.
5).

metastasis in this study. Thus, we try to analysis the ERK1/2,
p38, JNK1/2, and PI3K that are the FAK signals candidates, and
noticed the JNK signal was dramatically inhibited in the MCF-7
cells after apigenin treatment (Fig. 3a). The ERK1/2 and Akt were
also slightly decreased. In the meantime, the nuclear NFκB, c-Fos,
and c-Jun were downregulated (Fig. 3b), illustrate the FAK signal
can be inhibited by apigenin exposure 3 h.

Fig. 4 Low dose apigenin can influence the cytoskeleton. (a) The cytoskeleton regulator, including Ras, Rac-1, Cdc42, RhoA, and
RhoB were determined by western blot assay. The Ras and Rac-1 were markedly decreased, and the Cdc42 and RhoA were slight decreased
in MCF-7 cells after low dose apigenin exposure 3 h. (b) Phalloidin-PE stain in MCF-7 cells after low dose apigenin exposure
3 h, and then microscopy imaging to explore the cytoskeleton change. The cytoskeleton remodeling was clearly observed, like the
lamellipodia reduce in MCF-7 cells after the low dose apigenin exposure 3 h. Scale=50 μm.

Apigenin can regulate the cytoskeleton change to inhibit the
MCF-7 cells metastasis

As described above, low dose apigenin exposure 3 h down-regulate
MMP-2 and MMP-9 through FAK inhibition. On the other hand,
we also noticed cytoskeleton regulator influence in the low dose
apigenin exposure 3 h. The Ras, Rac-1, Cdc42, and RhoA were
the downstream of FAK associated with cytoskeleton remodeling.
Known the Ras and Rac-1 are related with lamellipodia, the Cdc42
is a regulator in filopodia, and the RhoA is associated with stress
fiber formation. Here, we observed the Ras and Rac-1 obviously
down regulation after low dose apigenin exposure 3 h as compared
with Cdc-42 and RhoA (Fig. 4a). After fluorescence microscopy
imaging, we also observed the lamellipodia was not obviously
express in MCF-7 cells after low dose apigenin exposure 3 h (Fig. 4b). The results suggest that apigenin inhibit breast cancer cell
metastasis is through FAK signal down regulation to cause matrix
metalloproteinase decreasing and cytoskeleton remodeling (Fig.5).

Fig. 5 The cartoon drawing is summarizing the effect of low dose
apigenin exposure 3 h in MCF-7 cells. Low dose apigenin can
trigger FAK deactivation in MCF-7 cells to reduce cell migration
and invasion through JNK signal downregulation. On the other hand, FAK deactivation also can influence the cytoskeleton
remolding through the cytoskeleton regulator inhibition.

Discussion

Tumor metastasis is the toughest problem in cancer therapy. Once
tumor cell metastasis, anticancer drugs could not achieve the
optimal efficiency, and the survival rate of patients with cancer also would reduced. Thus, avoid or reduce the phenomena of cancer
metastasis is the most important issue in the cancer research.
Cancer metastasis is the process of EMT. The epithelial cells lose
their cell polarity and cell-cell adhesion, and enhance its migratory
and invasive properties to become mesenchymal cell type. EMT
is also critical for development of many tissues and organs in the
developing embryo and numerous embryonic events [23]. Many
markers have been considered to be a fundamental event in EMT,
such as loss expression of E-cadherin, type IV collagen or laminin

In the meantime, increase the expression of N-cadherin, vimentin,
or fibronectin…ect [24]. EMT is also involved in cytoskeleton
remodeling [25] and the extracellular matrix (ECM) damaged [26].
Matrix metalloproteinases (MMPs) play key functions in degrade
and modify the ECM, gelatin-cleaving MMPs (MMP-2 and -9)
were the frequently molecule explored in cancer research with
metastasis. In this study, we verify the MMP-2 and -9 were down
regulation after low dose apigenin exposure 3h.

On the other hand, EMT is also a process of cytoskeleton change.
The actin cytoskeleton is a highly dynamic structure manifested
in all live cells. It is based on well-balanced of the local assembly
and disassembly of actin filaments. Rho GTPases are the mainly
responsible of transmitting signals from chemokine and growth
factor receptors and from adhesion receptors to the effector of actin
remodeling candidates, including RhoA, Rac1 and Cdc42. The
activity of RhoA during EMT is effects cell-cell adhesion, Ras and
Rac1 enhance the formation of protrusive membrane structures,
such as lamellipodia and Cdc42 can influence filopodia that are
rod-like extension consisting of tight bundled actin fibers which
penetrate into the surrounding environment originating from the
basis of lamellipodia. In this study, we noticed the Ras and Rac-
1 is decreased after low dose apigenin treatment implies the low
dose apigenin can decrease lamellipodia formation to reduce cell
motility.

Apigenin has been demonstrated strong inhibited HER2/neu
over-expressing breast cancer cells growth, but it was much less
effective in inhibiting growth of cells expressing basal levels of
HER2/neu [27]. Many studies were verifying the effect of apigenin
on breast cancer cell (Tab. 1). In these studies, 24-48 h exposure
time of apigenin treated in MCF-7 or MDA-MB-231 cells were
the most used to against the cell growth. The MAPK and PI3K/
Akt pathways were the most clarify in these studies. However,
FAK signal is the less described in breast cancer cell after apigenin
exposure. Here, we observed the cell growth could not be inhibited
in basal type breast cancer cell line, MDA-MB-231 in the 1-45 μg/
ml of apigenin exposure 24 or 48 h. Suggest that apigenin treated
in basal type breast cancer cell needs to take longer exposure time.
That is to say, low dose apigenin exposure 3 h in breast cancer
can achieve optimal effect is in the luminal type rather than basal
type. Nevertheless, low dose apigenin treatment is sufficient to
effect breast cancer cell cytoskeleton remodeling and decreasing
the matrix metalloproteinases expression through FAK signal to
reduce metastasis were verified in this study. In terms of cancer
therapy, low dose apigenin exposure short time may be the
potential for reduces lumina type cancer cell metastasis.

Tab. 1 The study of breast cancer and apigenin effect were described in below.

Acknowledgement

This study was supported by a grant from the Research Program
of Kaohsiung Armed Forces General Hospital (Project Number:101-29).